Morinda officinalis polysaccharide delays osteoarthritis mouse chondrocyte degeneration by modulating the glycolysis-pyroptosis axis via targeting the lncRNA XIST

doi:10.12122/j.issn.1673-4254.2025.12.02

Abstract

Abstract:

Objective To investigate the mechanism by which Morinda officinalis polysaccharide (MOP) delays osteoarthritis chondrocyte degeneration. Methods In primary cultures of chondrocytes from 4-week-old C57BL/6 mice, the effects of IL-1β and MOP treatment at different concentrations on cell viability were assessed with CCK-8 assay. The treated cells were examined for protein expressions of PKM2, caspase-1, and GSDMD using Western blotting and for XIST expression using fluorescence in situ hybridization (FISH). In IL-1β-induced mouse chondrocytes, the effects of MOP, transfection for XIST overexpression or knockdown, and MOP treatment after the transfection were tested by detecting mRNA levels of GluT1, HK2, PKM2, LDHA, PFKFB3, NLRP3, caspase-1, and GSDMD; flow cytometry, Western blotting, and toluidine blue staining were used to analyze chondrocyte apoptosis, expressions of glycolysis and pyroptosis regulators, and glycosaminoglycan expression. Results The second-passage chondrocytes showed good viability and positive collagen II staining. IL-1β induction caused degenerative morphological changes of the cells, decreased collagen II expression, and upregulated cellular expressions of PKM2, caspase-1, and GSDMD proteins. MOP treatment (especially at 4 mg/mL) significantly enhanced cell viability and reduced HK2, PKM2, caspase-1 and GSDMD expressions in IL-1β‑induced mouse chondrocytes. XIST was localized predominantly in the nuclei of the chondrocytes, and its expression increased significantly in IL-1β‑treated cells, and was attenuated by MOP treatment. XIST overexpression synergized with IL-1β to upregulate mRNA and protein expressions of glycolysis- and pyroptosis-related factors in the chondrocytes, and such effects were obviously attenuated by MOP. Conversely, XIST knockdown significantly inhibited chondrocyte apoptosis and glycosaminoglycan expression, and down-regulated glycolysis- and pyroptosis-related proteins. MOP treatment exhibited similar protective effects to XIST knockdown, and their combination significantly augmented these protective effects. Conclusions MOP mitigates IL-1β‑induced mouse chondrocyte degeneration by modulating glycolysis and pyroptosis via targeting XIST.

Key words: Morinda officinalis polysaccharide, osteoarthritis, degenerated chondrocytes, pyroptosis, glycolysis, lncRNA XIST

Changlong FU, Ruolan CHEN, Shiqi XU, Jinxin YOU, Qing LIN, Yanfeng HUANG. Morinda officinalis polysaccharide delays osteoarthritis mouse chondrocyte degeneration by modulating the glycolysis-pyroptosis axis via targeting the lncRNA XIST[J]. Journal of Southern Medical University, 2025, 45(12): 2541-2550.

Figures/Tables 8

Tab.1 Primer sequences for RT-qPCR

Gene	Primer sequences	Produet length (bp)
GLUT1	F:5'-ACCATCTTGGAGCTGTTCCG-3' R:5'-GCCTTCTCGAAGATGCTCGT-3'	131
HK2	F:5'-CCAGAGCATCCTCCTCAAGT-3' R:5'-GGTCTTCATAGCCACAAGTCATC-3'	173
PKM2	F:5'-AGGCTGCCATCTACCACTTG-3' R:5'-CCAGACTTGGTGAGCACGAT-3'	148
LDHA	F:5'-CAGACTTGGCTGAGAGCATAATG-3' R:5'-CCTTCACAACATCCGAGATTCC-3'	147
PFKFB3	F:5'-CAGGATCTTGTCCAACGCCT-3' R:5'-AGGCTAGTAGGCAGCGTAGA-3'	103
NLRP3	F:5'-TCCCAGACACTCATGTTGCC-3' R:5'-GTCCAGTTCAGTGAGGCTCC-3'	116
Caspase-1	F:5'-ACTGACTGGGACCCTCAAGT-3' R:5'-GCAAGACGTGTACGAGTGGT-3'	111
GSDMD	F:5'-TTAATTGAGGCGGCAGACTTC-3' R:5'-CGATGTTCACCAGTATCTGTTGT-3'	109
GAPDH	F:5'-TGGAAAGCTGTGGCGTGATG-3' R:5'-TACTTGGCAGGTTTCTCCAGG-3'	189

Fig.1 Primary culture, induction, and identification of mouse chondrocytes. A: Morphology of primary chondrocytes. B: Morphology of the second-passage chondrocytes. C: Positive expression of collagen II protein. D: Negative expression of collagen II protein. E: Morphology of chondrocytes induced by IL-1β. F: IL-1β induces positive expression of collagen II protein.

Fig.3 Effect of different concentrations of MOP on protein expressions of PKM2, caspase-1 and GSDMD in mouse chondrocytes. Data are presented as Mean±SD (n=3). *P<0.05 vs Control group, #P<0.05 vs IL-1β group.

Fig.5 Effect of MOP on mRNA levels of GLUT1, HK2, PKM2, LDHA, PFKFB3, NLRP3, caspase-1, and GSDMD in mouse chondrocytes overexpressing XIST with IL-1β treatment. Data are presented as Mean±SD (n=3). *P<0.05 vs Control group; #P<0.05 vs IL-1β group; ▲P<0.05 vs IL-1β+oe-XIST group.

Fig.6 Effect of MOP on protein expressions of HK2, PKM2, PFKFB3, NLRP3, caspase-1, and GSDMD in mouse chondrocytes overexpressing XIST with IL-1β treatment. Data are presented as Mean±SD (n=3). *P<0.05 vs Control group; #P<0.05 vs IL-1β group; ▲P<0.05 vs IL-1β+oe-XIST group.

Fig.7 Effect of MOP on apoptosis in mouse chondrocytes with XIST knockout and IL-1β treatment. Data are presented as Mean±SD (n=3). *P<0.05 vs Control group; #P<0.05 vs IL-1β group; ▲P<0.05 vs IL-1β+sh-XIST group.

Fig.8 Effect of MOP on glycosaminoglycan expression in mouse chondrocytes with XIST knockout and IL-1β treatment (Scale bar=100 μm).

Fig.9 Effect of MOP on protein expressions of HK2, PKM2, PFKFB3, NLRP3, caspase-1, and GSDMD in mouse chondrocytes with XIST knockout and IL-1β treatment. Data are presented as Mean±SD (n=3). *P<0.05 vs Control group; #P<0.05 vs IL-1β group; ▲P<0.05 vs IL-1β+sh-XIST group.

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